Medicine by Design funded the following projects through New Ideas Grants in spring 2016:
Regeneration of auditory neurons for the treatment of hearing loss
Project PI: Alain Dabdoub
Sunnybrook Research Institute
Project Description: Hearing loss is the most common sensory disability. It affects 0.3 per cent of newborns, 5 per cent of people under age 45, and 50 per cent by age 70, and impacts 400 million people worldwide. In the mammalian cochlea, primary auditory neurons (PAN) are responsible for transmitting auditory information from the mechanosensory hair cells in the cochlea to the brainstem. PANs do not regenerate; thus, when they are lost or damaged due to noise exposure or aging, their loss is prevalent and permanent, and leads to hearing impairment. The development of methodologies that could be used to induce the regeneration of auditory neurons in a damaged ear, therefore, has significant implications for future advances in the treatment of hearing loss. The potential clinical value of the regenerative therapy in the amelioration of hearing impairment is tremendous. The approach proposed in this project for hearing loss treatment is to use gene therapy, which offers rigorous methods for characterization of functional recovery. Several cochlear gene therapy studies have focused on preserving PANs by gene transfection of neurotrophins, showing the feasibility of gene therapy for protection of PANs and regrowth of neurites to cochlear hair cells peripherally. Direct cell reprograming is an emerging area of regenerative medicine and, as proof of principle, Dr. Dabdoub and his team have successfully induced neurons by overexpression of transcription factors in non-sensory epithelial cells in the inner ear. Herein, they propose to convert endogenous glial cells that normally surround PANs and survive after neuron loss, into regenerated PANs. The ultimate goal is to induce PANs in human ears to enable functional innervation to both the cochlear nucleus in the central nervous system and hair cells or cochlear implants in the periphery. The induction of even a small number of neurons in a damaged ear has significant implications for clinical research related to cochlear implants and hearing restoration, because as few as 10 per cent (3,500) of the congenital normal number (35,500) of PANs could result in auditory sensation.
In vivo visualization of hESC-derived β-cell stem differentiation, growth and function
Project PI: Cristina Nostro
McEwen Centre for Regenerative Medicine & Toronto General Research Institute, University Health Network
Department of Physiology, University of Toronto
Co-PI: Herbert Gaisano
Department of Physiology, University of Toronto
Project Description: Diabetes is a major cause of mortality and morbidity worldwide, with increasing rates, inflicting unsustainable costs to healthcare. Major contributors to diabetes are loss of β-cell mass from autoimmune destruction (type-1 diabetes) and β-cell function from metabolic perturbation (type-2 diabetes). The two most effective treatments for diabetes are insulin replacement (discovered in Toronto a century ago) and β-cell replacement by islet transplantation (also a Canadian discovery). The latter, seemingly ideal, is severely restricted by organ availability (two to three pancreases are needed per patient) and immunosuppression. To surmount these obstacles, a third option exists — developing an “infinite” supply of insulin-producing cells. This is now made possible by recent innovations in stem cell biology demonstrating the feasibility to generate insulin-producing cells from human embryonic stem cells (hESC). Dr. Nostro has made great strides in this area, and is one of a handful in the world that have perfected driving hESCs towards pancreatic progenitors (PPs), which when transplanted into type-1 diabetes rodents generate all lineages of the pancreas. Using a modified version of a published protocol, the PPs can be differentiated in vitro into β-like cells. However, the major limiting factor for a more rapid progress has been the use of conventional readouts: histology to verify differentiation; generating very large population of differentiated cells over many months sufficient for insulin secretion assays and in vivo implants to restore glycemic control in diabetic rodent models. To surmount these huge obstacles and to be first to finish this “race” requires an innovative strategy, which is to deploy an in vivo observation chamber that can track β-cell differentiation, mass and secretory function. Dr Gaisano (co-investigator on project), a global lead in islet function biology, has developed a powerful in vivo imaging assay (with a 2-photon microscope) capable of longitudinal observation of implanted human islet grafts in the anterior chamber of the eye of severe combined immunodeficient (SCID) mice. In this project, Dr. Nostro’s hESC-derived β-like cells engineered to express insulin granule fluorophores will be implanted into the eye of diabetic SCID mice. In vivo stimulation of whole islet exocytosis (increased insulin granule fluorescence) will track both β-cell mass and insulin secretory capacity; and consequent effects on whole animal glucose homeostasis concurrently assayed. They will survey treatments with novel drug compounds to see how and which ones improve/maintain β-cell differentiation, growth and secretion.
The role of architecture and geometry in stem cell fate choice
Project PI: Tom Waddell
University Health Network
Project Description: The physical microenvironment is a key factor influencing fate choice of pluripotent and progenitor cell populations. Little is understood about the mechanism by which specific mechanical forces affect stem cell differentiation. During development, lung and pancreas are derivatives of endoderm. Human embryonic stem cells (hESCs) can be differentiated in vitro towards definitive endoderm through exposure to a series of growth factors and small molecule agonists and inhibitors to putative multipotent lung progenitors and multipotent pancreatic progenitors. In both lung and pancreas, while some mature phenotypes are seen in in vitro cultures, there is a clear lack of organization with inappropriate cell types juxtaposed. The focus of the directed differentiation field has been on the refinement of the schedule of growth factors and small molecules. Yet the result has only been to create mixed populations of limited yield and purity. Dr. Waddell’s team hypothesizes that to achieve more clinically relevant populations, chemically directed differentiation protocols must be augmented with architecturally relevant microenvironments that mimic in vivo condition. In addition to providing fundamental knowledge on the effect of architecture on stem cell differentiation, this approach will enable generation of populations with greater purity with the potential of scale up. This has wide implications in regenerative medicine and specifically in tissue engineering applications currently limited by cell number and purity. For example, recellularization of decellularized tissues is a major avenue of research in the field. Deficiencies in the number and type of cells is one of the major stumbling blocks to advancement in this area. In the shorter term, architecturally enhanced differentiation protocols will be used for disease modelling, and drug screening applications using patient-specific induced pluripotent stem cell (iPSC)-derived cells. As a proof of principle they will examine cystic fibrosis where the clinical need for drug screening models is great, and especially, the study of pancreatic ductal cells would be very novel.